We are interested in developing tests for effects of metal exposures upon human sperm function. We propose to follow up findings from our current prospective study of males of couples undergoing in vitro fertilization (IVF) that 42/95 subjects had elevated blood and semen Pb2+ (greater than 40 microgram/dcl), and that rates of female partner oocyte fertilization in IVF correlated inversely (r=-448, P less than 0.001) with these Pb+2 levels. In this cohort, seminal plasma Pb+2 correlated inversely (r=-.523, P less than 0.01) with the ability of sperm to undergo a progesterone-stimulated acrosome reaction (PSAR). When visualized with probes for non-nuclear progesterone receptor (NNPR) and mannose lectin (ML), the principal site of Pb+2 blockade lay at an early step of the PSAR. Capacitation, as determined by expression levels of NNPR and ML was affected, but slightly compared to controls, but the relocation of NNPR and ML from the anterior sperm head to the sperm equatorial segment (which normally precedes Ca+2 influx through a voltage-gated calcium channel and in turn triggers fusion of acrosomal and cell membranes) was largely arrested. Blood Pb+2 was unrelated to hormone levels, suggesting Pb+2 effects were not mediate through endocrine disruptors. As adding either micromolar Pb+2 or specific inhibitors of delayed-rectifier voltage-gated K+ channels from human and rat testes cDNA also suggested such channels exist in human sperm. Polymorphisms found in Northern hybridizations suggest that individuals who differed in their productive response to similar [Pb+2} challenge may have variant K+ channel isoforms. We will complete sequencing of the human sperm delayed-rectifier voltage-gated K+ channel, and will attempt to clone the human form of the hamster Ca2+-activated K+ channel. We will develop DNA probes to characterize alternatively spliced K+ channels. We will make a second prospective study of 150 male IVF patients to confirm our findings and seek to identify further environmental risk factors for Pb2+ effects on human sperm function, correlating levels of expression of variant K+ channel mRNAs in sperm with blood and seminal plasma [Pb2+] and with sperm NNPR expression and the PSAR. We will also investigate a possible mechanism for Pb2+'s reproductive effects on male sperm, that it affects sperm K+ channel function or interferes with early signal transduction after membrane depolarization. We will use metal-ion-sensing fluorescent probes, and specific inhibitors to probe the effects of PB2+ upon ion fluxes. We will employ phospholipase C assays and competition assays to probe the effects of Pb2+ upon inositol phosphate secondary messages. The goal of this application is to determine the mechanisms of injury to the male reproductive tract resulting from environmental and occupational exposure to heavy and transition metal ions. The focus being on the effects of lead exposure on acrosome reaction insufficiency and male infertility. The strength application include the expertise and experience of the principal investigator Dr. Benoff and her research team, the novel approach to analysis of the problem.